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[0002] This invention was made with government support under
09-CRHF-0-6055 awarded by the USDA/CSREES. The government has certain
rights in the invention.

Claims

1. A collection of at least two of isolated polynucleotide molecule
species selected from the group consisting of (1) an isolated
polynucleotide comprising at least 12 consecutive nucleotides surrounding
position of 1296 of SEQ ID NO:1; (2) an isolated polynucleotide
comprising at least 12 consecutive nucleotides surrounding position of
213 of SEQ ID NO:2; (3) an isolated polynucleotide comprising at least 12
consecutive nucleotides surrounding position of 8504 of SEQ ID NO:3; (4)
an isolated polynucleotide comprising at least 12 consecutive nucleotides
surrounding position of 154963 of SEQ ID NO:4; (5) an isolated
polynucleotide comprising at least 12 consecutive nucleotides surrounding
position of 577 of SEQ ID NO:5; (6) an isolated polynucleotide comprising
at least 12 consecutive nucleotides surrounding position of 23 of SEQ ID
NO:6: (7) an isolated polynucleotide comprising at least 12 consecutive
nucleotides surrounding position of 11646 of SEQ ID NO:6; and (8) an
isolated polynucleotide comprising at least 12 consecutive nucleotides
surrounding position of 12195 of SEQ ID NO:7.

2. The collection according to claim 1, comprising at least three
species.

3. The collection of claim 2, comprising all eight species.

4. The collection of claim 1, wherein the nucleotide species are on a
solid support.

5. The collection of claim 1, wherein the nucleotide species are arranged
in an addressable array.

6. The collection of claim 4, wherein the nucleotide species are arranged
in an array on a solid support.

7. The collection of claim 6, wherein the array is made of silicon,
glass, plastic, or paper.

8. The method of claim 6, wherein the array is formed into wells on
plates, slides, sheets, membranes, fibers, chips, dishes, and beads.

9. The collection of claim 6, wherein array is treated, coated or
derivatized to facilitate the immobilization of the nucleotide molecules.

10. A method for genotyping a bovine cell, comprising obtaining a nucleic
acid sample from said cell and determining the identity of the nucleotide
of eight SNP positions in the cell, wherein the eight SNP positions are
(1) position 1296 of SEQ ID NO:1; (2) position 213 of SEQ ID NO:2; (3)
position 8504 of SEQ ID NO:3; (4) position 154963 of SEQ ID NO:4; (5)
position 577 of SEQ ID NO:5; (6) position of 23 SEQ ID NO:6; (7) position
11646 of SEQ ID NO:6; and (8) position 12195 of SEQ ID NO:7, the method,
comprising (1) Determining the identity of a nucleotide at each of the
eight SNP positions, and (2) comparing the identity to the nucleotide
identity at a corresponding position of in SEQ ID NOs: 1-7, respectively.

11. The method according to claim 10, wherein the bovine cell is an adult
cell, an embryo cell, a sperm, an egg, a fertilized egg, or a zygote.

12. The method according to claim 10, wherein the identity of the
nucleotide is determined by sequencing or a relevant fragment of the
respective gene isolated from the cell.

13. A method according to claim 12, wherein relevant fragment of the
respective gene is isolated from the cell via amplification by the
polymerase chain reaction (PCR) of genomic DNA of the cell, or by RT-PCR
of the mRNA of the cell.

14. A method according to claim 10, wherein both copies of the respective
gene in the cell are genotyped.

15. A method for progeny testing of cattle, the method comprising
collecting a nucleic acid sample from said progeny, and genotyping said
nucleic sample according to claim 10.

16. A method for selectively breeding of cattle using a multiple
ovulation and embryo transfer procedure (MOET), the method comprising
superovulating a female animal, collecting eggs from said superovulated
female, in vitro fertilizing said eggs from a suitable male animal,
implanting said fertilized eggs into other females allowing for an embryo
to develop, and genotyping said developing embryo according to claim 10,
and terminating pregnancy if said developing embryo does not all have a
corresponding desired polymorphic nucleotide as shown in Table 1A.

17. A method according to claim 16, wherein pregnancy is terminated if
the embryo is not homozygous with regard to all of the corresponding
desired polymorphic nucleotide.

18. A method for selectively breeding dairy cattle, comprising selecting
a bull that is homozygous with regard to all desired polymorphic
nucleotides as shown in Table 1A and using its semen for fertilizing a
female animal.

19. A method according to claim 18, wherein the female animal is in vitro
fertilized.

20. A method according to claim 18, wherein MOET procedure is used.

21. A method according to claim 18, wherein said female animal is also
homozygous with regard to all desired polymorphic nucleotides as shown in
Table 1A.

22. A method for testing a dairy cattle for its fertility, comprising
genotyping its cells according to claim 13, wherein a cattle homozygous
with regard to all desired polymorphic nucleotides as shown in Table 1A
indicates that the cattle has fertility rate.

Description

CROSS-REFERENCE TO RELATED APPLICATION

[0001] This is a continuation application of U.S. application Ser. No.
12/637,753 filed on Dec. 15, 2009, claiming priority to U.S. Patent
Application 61/122,524, filed on Dec. 15, 2008, the entire disclosure of
which is incorporated herein by reference.

FIELD OF THE INVENTION

[0003] The present invention relates to methods of genetic testing of
cattle using molecular genetic methods by assaying for the presence of at
least one genetic marker which is indicative of fertility or embryonic
survival.

BACKGROUND OF THE INVENTION

[0004] Dairy cows are significant investments for dairy farmers, and
enormous efforts, such as animal breeding and artificial insemination,
have been and continue to be invested in ensuring that the animals have
high and sustained productivity, and that the milk produced is of high
quality. About 50 quantitative trait loci (QTL) affecting milk production
traits have been identified (Bagnato et al., 2008; Lipkin et al., 2008).
The dairy cattle genome has been significantly restructured over the past
30 years due to intense selection for production traits.

[0005] Such restructuring of the dairy cattle genome over the past 30
years due to intense selection for production traits may have resulted in
a hitchhiking effect on a large number of loci adversely affecting
fertilization rate and embryo survival, leading to dairy cattle genotypes
that are suboptimal for reproductive competence (Royal et al., 2000;
Lucy, 2001). The decrease in dairy cattle fertility is a worldwide
problem and a major cause of economic loss and cow culling in the global
dairy herd.

[0006] Many reasons account for this reduced reproductive efficiency, but
the most important component seems to be a reduction in embryo survival
rate from over 80% twenty years ago to less than 50% today. There appears
to be an important genetic basis for this decline (Veerkamp and Beerda,
2007); so genetic approaches may help alleviate this problem. As such,
there is an urgent need to identify the genetic factors responsible for
the decline in embryo survival rate.

[0007] Previously the present inventor has demonstrated the effectiveness
of the candidate pathway approach in choosing candidate genes affecting
milk production traits (Leonard et al., 2005; Cobanoglu et al., 2006;
Khatib et al., 2007a,b; Khatib et al., 2008a; Wang et al., 2008).
Recently an in vitro fertilization (IVF) experimental system in cattle
has been demonstrated that enables the association of single nucleotide
polymorphisms (SNPs) in candidate genes with fertilization rate and
embryo survival. Using this system, two genes: fibroblast growth factor 2
(FGF2) and signal transducer and activator of transcription 5 (STAT5A)
were found to be significantly associated with variation in fertilization
and embryo survival rates (Khatib et al., 2008a,b). These two genes were
chosen from the interferon-tau (IFNT) and placental lactogen (PL) signal
transduction pathway.

[0008] Interferon-.tau. (IFNT) is a major product of ovine and bovine
conceptuses during the period before the trophoblast makes firm
attachment to the uterine wall and begins to form a placenta. Its primary
function is in preventing a return to ovarian cyclicity and hence
ensuring the pregnancy to continue, although it undoubtedly has other
roles in ensuring receptivity of the maternal endometrium.

[0010] Identifying additional genetic factors that show association with
fertilization rate or embryo survival rate would enable selection or
breeding programs that reduce the frequency of deleterious alleles at
these loci by marker- or gene-assisted selection, preventing further
decline or even improving reproductive status of the global dairy herd.

[0011] Furthermore, a plurality of or multiple genes are likely more
reliable than a single gene or SNP in predicting high fertility or
enhanced embryo survival.

SUMMARY OF THE INVENTION

[0012] The present inventor investigated the effects of various genes of
the IFNT signaling pathway and discovered that several of these genes
comprise SNPs that are correlated with increased fertilization rate, or
embryo survival rate, or both, and these SNPs may be used in breeding
programs or other cattle testing or selection programs for cattle with
improved fertility, more specifically for increased pregnancy rate in
cattle. Accordingly, in one embodiment, the present invention provides a
collection, or an array, of at least two of isolated polynucleotide
molecule species selected from the group consisting of (1) an isolated
polynucleotide comprising at least 12 consecutive nucleotides surrounding
position of 1296 of SEQ ID NO:1; (2) an isolated polynucleotide
comprising at least 12 consecutive nucleotides surrounding position of
213 of SEQ ID NO:2; (3) an isolated polynucleotide comprising at least 12
consecutive nucleotides surrounding position of 8504 of SEQ ID NO:3; (4)
an isolated polynucleotide comprising at least 12 consecutive nucleotides
surrounding position of 154963 of SEQ ID NO:4; (5) an isolated
polynucleotide comprising at least 12 consecutive nucleotides surrounding
position of 577 of SEQ ID NO:5; (6) an isolated polynucleotide comprising
at least 12 consecutive nucleotides surrounding position of 23 of SEQ ID
NO:6; (7) an isolated polynucleotide comprising at least 12 consecutive
nucleotides surrounding position of 11646 of SEQ ID NO:6; and (8) an
isolated polynucleotide comprising at least 12 consecutive nucleotides
surrounding position of 12195 of SEQ ID NO:7. Preferably, the collection
comprises at least three, at least four, at least five, at least six, or
at least seven species described above. More preferably, the collection
comprises all eight species.

[0013] In another embodiment, the present invention provides a method for
genotyping a bovine cell, comprising obtaining a nucleic acid sample from
said cell and determining the identity of the nucleotide of eight SNP
positions in the cell, wherein the eight SNP positions are (1) position
1296 of SEQ ID NO:1; (2) position 213 of SEQ ID NO:2; (3) position 8504
of SEQ ID NO:3; (4) position 154963 of SEQ ID NO:4; (5) position 577 of
SEQ ID NO:5; (6) position of 23 SEQ ID NO:6; (7) position 11646 of SEQ ID
NO:6; and (8) position 12195 of SEQ ID NO:7, the method, comprising (1)
determining the identity of a nucleotide at each of the eight SNP
positions, and (2) comparing the identity to the nucleotide identity at a
corresponding position of in SEQ ID NOs: 1-7, respectively. In preferred
embodiments, the method according to the present invention is used to
test an adult bovine cell, an embryonic bovine cell, a bovine sperm, a
bovine egg, a fertilized bovine egg, or a bovine zygote. In one
embodiment, both copies of the respective gene in the cell are genotyped.

[0014] In another embodiment, the present invention provides a method for
selectively breeding of cattle using a multiple ovulation and embryo
transfer procedure (MOET), the method comprising super-ovulating a female
animal, collecting eggs from said superovulated female, in vitro
fertilizing said eggs from a suitable male animal, implanting said
fertilized eggs into other females allowing for an embryo to develop, and
genotyping said developing embryo as described above, and terminating
pregnancy if said developing embryo does not all have a corresponding
desired polymorphic nucleotide as shown in Table 1A.

DESCRIPTION OF THE DRAWINGS

[0015] FIG. 1 shows the partial sequence of the UTMP gene (SEQ ID NO:1)
where the relevant SNP position is noted.

[0016] FIG. 2 shows the partial sequence of the STAT1 gene (SEQ ID NO:2)
where the relevant SNP position is noted.

[0017] FIG. 3 shows the partial sequence of the OPN gene (SEQ ID NO:3)
where the relevant SNP position is noted.

[0018] FIG. 4 shows the partial sequence of the GHR gene (SEQ ID NO:4)
where the relevant SNP position is noted.

[0019] FIG. 5 shows the partial sequence of the POU1F1 gene (SEQ ID NO:5)
where the relevant SNP position is noted.

[0020] FIGS. 6A, 6B, 6D and 6C together show the partial sequence of the
FGF2 gene (SEQ ID NO:6) where the two relevant SNP positions at positions
23 and 11646 are noted.

[0021] FIG. 7 shows the partial sequence of the STAT5A gene (SEQ ID NO:7)
where the relevant SNP position is noted.

DETAILED DESCRIPTION OF THE INVENTION

[0022] It has now been found that many genes encoding proteins of the IFNT
signaling pathway contain single nucleotide polymorphisms (SNPs), and
certain of these alleles correspond to increased fertilization rate, or
embryonic survival rate, or both, in dairy cattle, and the beneficial
effects of these alleles are additive. Specifically, it has been
discovered that SNPs exist in the following genes: growth hormone
receptor (GHR), osteopontin (OPN/SPP1), POU1F1, signal transducer and
activator of transcription (STAT1), signal transducer and activator of
transcription (STAT5A), bovine uterine milk protein (UTMP), and
fibroblast growth factor 2 (FGF2).

[0029] These and other references cited herein are all incorporated by
reference in their entirety.

[0030] POU1F1 is a member of the tissue specific POU (Pit, Oct, Unc)
homeobox transcription factor DNA binding protein family that is found in
all mammals studied so far (Bastos et al., 2006; Ingraham et al., 1988;
Ingraham et al., 1990). The pituitary specific expression of POU1F1 is
required for the activation of growth hormone (GH), prolactin (PRL), and
thyroid stimulating hormone (TSH) (Li et al., 1990). These genes are
involved in a variety of signaling pathways that are important for many
developmental and physiological processes, including pituitary gland
development (Li et al., 1990. Mullis, 2007), mammary gland development
and growth (Svennersten-Sjaunja and Olsson, 2005), milk protein
expression (Akers, 2006), and milk production and secretion
(Svennersten-Sjaunja and Olsson, 2005). Moreover, binding of GH and PRL
to their receptors on the cell membrane triggers a cascade of signaling
events including the JAK/STAT pathway, which has been shown to be
required for adult mammary gland development and lactogenesis (Liu et
al., 1997).

[0031] Several genes in the same pathway of POU1F1 have been reported to
be associated with different milk production and health traits. For
example, growth hormone receptor (GHR) has shown associations with milk
yield and composition (Viitala et al., 2006). Also, the signal transducer
and activator of transcription 1 (STAT1) and osteopontin (OPN) genes have
been shown to have significant effects on milk yield and milk protein and
fat yields in Holstein dairy cattle (Cobanoglu et al., 2006; Leonard et
al., 2005: Schnabel et al., 2005). The uterine milk protein (UTMP) is
another gene in the pathway of POU1F1 that has been found to be
associated with productive life in dairy cattle (Khatib et al., 2007b).

[0032] The FGF2 regulates the trophectoderm expression of
interferon-.tau., a key member of the signal transduction pathway
involved in milk production (Ocon-Grove et al., 2007). Bovine FGF2 is
mapped to chromosome 17, with 3 exons and a total length of over 55 kb;
it is expressed by the endometrium throughout the estrous cycle and early
pregnancy (Michael et al., 2006).

[0033] The signal transducer and activator (STAT) proteins are known to
play an important role in cytokine signaling pathways. STAT proteins are
transcription factors that are specifically activated to regulate gene
transcription when cells encounter cytokines and growth factors, hence
they act as signal transducers in the cytoplasm and transcription
activators in the nucleus (Kisseleva et al., 2002). In mammals, STATs
comprise a family of seven structurally and functionally related
proteins: STAT1, STAT2, STAT5, STAT4, STAT5a and STAT5b, STAT6 (Darnell,
1997). The seven mammalian STAT proteins range in size from 750 to 850
amino acids. The chromosomal distribution of these STATs, as well as the
identification of STATs in more primitive eukaryotes, suggest that this
family arose from a single primordial gene (Chen et al., 1998). In
addition, STATs share a number of structurally and functionally conserved
domains.

[0034] The STAT5 protein is also known as the mammary gland factor. This
protein was initially identified in the mammary gland as a regulator of
milk protein gene expression (Watson, 2001). STAT5A is a member of the
interferon-tau (IFN-tau) and placental lactogen (PL) signaling pathway,
which is involved in signal transduction within a variety of cells,
including the uterus and mammary epithelial cells. The uterus is exposed
to IFN-tau and PL, as well as many others hormones including estrogen,
progesterone, and placental growth hormone. The PL stimulates the
formation of STAT5 homodimers, which in turn induce the transcription of
the bovine uterine milk protein (UTMP) and osteopontin (OPN) genes
(Spencer and Bazer, 2002; Stewart et al., 2002; Spencer and Bazer, 2004).
In previous studies, the present inventor showed that the UTMP (Khatib et
al., 2007a) and OPN (Leonard et al. 2005; Khatib et al. 2007b) genes have
surprisingly strong effects on milk production and health traits in
cattle. Furthermore, the present inventor showed that STAT1--also member
of the IFN-tau and PL signal transduction pathway--is associated with
milk composition and health traits (Cobanoglu et al., 2006).

[0035] Studies in mouse have shown that STAT5A is involved in both milk
production and fertility; Stat5 knockout female mice fail to lactate
(Miyoshi et al., 2001). Also, it has been shown that disruption of Stat5
leads to infertility in females as a result of small-sized or a lack of
corpora lutea (Teglund et al., 1998). Because the primary source of
progesterone is the corpora lutea of the ovary, lack of development of
corpora lutea would have significant effects on the establishment of
pregnancy.

[0036] Polymorphisms at the nucleic acid level may provide functional
differences in the genetic sequence, through changes in the encoded
polypeptide, changes in mRNA stability, binding of transcriptional and
translation factors to the DNA or RNA, and the like. Polymorphisms are
also used to detect genetic linkage to phenotypic variation.

[0037] One type of polymorphism, single nucleotide polymorphisms (SNPs),
has gained wide use for the detection of genetic linkage recently. SNPs
are generally biallelic systems, that is, there are two alleles that an
individual may have for any particular SNP marker. In the instant case,
the SNPs are used for determining the genotypes of the POU1F1 gene, which
are found to have strong correlation to longevity and milk production
traits.

[0038] Through the following testing and analysis, it has been established
that certain alleles of the SNPs shown in Table 1 correspond to increased
fertilization rate, or embryonic survival rate, or both, in dairy cattle,
and the beneficial effects of these alleles are additive.

[0039] Gene Selection and Genotyping. The genes POU1F1, GHR, STAT5A, OPN,
UTMP, STAT1, and FGF2 were chosen for association tests with fertility
traits because they are members of the IFNT and PL/POU1F1 pathway.
Genotyping of these genes was performed as described in the literature
(Table 1) except for GHR, for which primers, GHR-F
CTTTGGAATACTTGGGCTAGCAGTGACA''A''TAT (SEQ ID NO:8) and GHR-R
GTCTCTCTGTGGACACAACA (SEQ ID NO:9) were used to amplify a 230-bp genomic
fragment. The original T nucleotide at position -4 of the SNP was mutated
to an A nucleotide in the forward primer to create an Ssp/recognition
site. Restriction enzyme digestions were carried out according to the
manufacturer's instructions.

[0040] Fertility Data Collection.

[0041] Ovaries from mature cows were collected from a local abattoir and
immediately used in the IVF experiments as described in Khatib et al.
(2008a,b). Briefly, oocytes were aspirated from antral follicles (>2-6
mm) and immediately incubated in maturation medium. On average, 12
oocytes were aspirated from each ovary. On day 2 (d 2), oocytes were
fertilized with frozen-thawed percoll-separated semen that had been
adjusted to a final concentration of 1 million sperm/ml. Fertilization
rate was calculated as the number of cleaved embryos at 48 h post
fertilization out of total number of oocytes exposed to sperm. Survival
rate of embryos was calculated as the number of blastocysts on d 7 of
development out of the number of total embryos cultured. Viability was
determined as a function of the embryo's ability to attain the
morphological stage of blastocyst on d 7 of development. Embryos that
failed to show cellular compaction (morula stage) on d 5 or d 6 were
considered nonviable. Therefore, only embryos exhibiting adequate
compaction followed by the formation of a blastocoele on d 7 were
considered viable. Ovaries from which fewer than 4 oocytes were harvested
were discarded and not further analyzed. A total of 7,413 fertilizations
were performed using oocytes from a total of 504 ovaries and semen from
10 different bulls.

[0042] Association of Individual Genes with Fertilization and Survival
Rates.

[0043] Associations of individual genes with fertilization and survival
rates were analyzed using the following logistic regression model:

is the natural logarithm of odds of survival rate or fertilization rate,
.beta..sub.0 is a general constant, .beta..sub.1j is the fixed effect
associated with the j.sup.th bull (Bull.sub.j; and .beta..sub.2k is the
genotype effect associated with the k.sup.th genotype (Genotype.sub.k) of
the gene analyzed. This model was fitted by Maximum Likelihood approach.
Association between the gene and survival/fertilization rate was tested
using a Likelihood Ratio Test (LRT).

[0044] Association of Candidate Genes with Embryonic Survival.

[0045] The GHR, STAT5A, UTMP, FGF2 SNP11646, FGF2 SNP23, and STAT1 genes
showed considerable associations with embryonic survival rate (Table 2).
For GHR, the survival rate of embryos produced from AA ovaries was 9%
higher than that of embryos produced from TT ovaries. For STAT5A, CC
ovaries showed 9% and 8% higher survival rates than that of GG and GC
ovaries, respectively. The UTMP gene showed 6% survival rate differences
between AA and GG genotypes (Table 2). SNP 11646 and SNP23 of FGF2 showed
differences of 7% each between genotypes GG and AA and between GG and TT,
respectively. For STAT1, although not statistically significant, TT
genotype was associated with a 4% increase in survival rate compared to
GG genotype.

[0048] In the context of the present invention, the provided sequences
also encompass the complementary sequence corresponding to any of the
provided polymorphisms. In order to provide an unambiguous identification
of the specific site of a polymorphism, the numbering of the original
nucleic sequences in the GenBank is shown in the figures and is used.

[0049] The present invention provides nucleic acid based genetic markers
for identifying bovine animals with superior fertility and survival
traits. In general, for use as markers, nucleic acid fragments,
preferably DNA fragments, will be of at least 12 nucleotides (nt),
preferably at least 15 nt, usually at least 20 nt, often at least 50 nt.
Such small DNA fragments are useful as primers for the polymerase chain
reaction (PCR), and probes for hybridization screening, etc.

[0050] The term primer refers to a single-stranded oligonucleotide capable
of acting as a point of initiation of template-directed DNA synthesis
under appropriate conditions (i.e., in the presence of four different
nucleoside triphosphates and an agent for polymerization, such as, DNA or
RNA polymerase or reverse transcriptase) in an appropriate buffer and at
a suitable temperature. The appropriate length of a primer depends on the
intended use of the primer but typically ranges from 15 to 30
nucleotides. Short primer molecules generally require cooler temperatures
to form sufficiently stable hybrid complexes with the template. A primer
need not reflect the exact sequence of the template but must be
sufficiently complementary to hybridize with a template. The term primer
site, or priming site, refers to the area of the target DNA to which a
primer hybridizes. The term primer pair means a set of primers including
a 5' upstream primer that hybridizes with the 5' end of the DNA sequence
to be amplified and a 3', downstream primer that hybridizes with the
complement of the 3' end of the sequence to be amplified.

[0051] The term "probe" or "hybridization probe" denotes a defined nucleic
acid segment (or nucleotide analog segment) which can be used to identify
by hybridization a specific polynucleotide sequence present in samples,
said nucleic acid segment comprising a nucleotide sequence complementary
of the specific polynucleotide sequence to be identified. "Probes" or
"hybridization probes" are nucleic acids capable of binding in a
base-specific manner to a complementary strand of nucleic acid.

[0052] An objective of the present invention is to determine which
embodiment of the polymorphisms a specific sample of DNA has. For
example, it is desirable to determine whether the nucleotide at a
particular position is A or C. An oligonucleotide probe can be used for
such purpose. Preferably, the oligonucleotide probe will have a
detectable label, and contains an A at the corresponding position.
Experimental conditions can be chosen such that if the sample DNA
contains an A, they hybridization signal can be detected because the
probe hybridizes to the corresponding complementary DNA strand in the
sample, while if the sample DNA contains a G, no hybridization signal is
detected.

[0053] Similarly, PCR primers and conditions can be devised, whereby the
oligonucleotide is used as one of the PCR primers, for analyzing nucleic
acids for the presence of a specific sequence. These may be direct
amplification of the genomic DNA, or RT-PCR amplification of the mRNA
transcript of the POU1F1 gene. The use of the polymerase chain reaction
is described in Saiki et al. (1985) Science 230:1350-1354. Amplification
may be used to determine whether a polymorphism is present, by using a
primer that is specific for the polymorphism. Alternatively, various
methods are known in the art that utilize oligonucleotide ligation as a
means of detecting polymorphisms, for examples see Riley et al (1990)
Nucleic Acids Res. 18:2887-2890; and Delahunty et al (1996) Am. J. Hum.
Genet. 58:1239-1246. The detection method may also be based on direct DNA
sequencing, or hybridization, or a combination thereof. Where large
amounts of DNA are available, genomic DNA is used directly.
Alternatively, the region of interest is cloned into a suitable vector
and grown in sufficient quantity for analysis. The nucleic acid may be
amplified by PCR, to provide sufficient amounts for analysis.

[0054] Hybridization may be performed in solution, or such hybridization
may be performed when either the oligonucleotide probe or the target
polynucleotide is covalently or noncovalently affixed to a solid support.
Attachment may be mediated, for example, by antibody-antigen
interactions, poly-L-Lys, streptavidin or avidin-biotin, salt bridges,
hydrophobic interactions, chemical linkages, UV cross-linking baking,
etc. Oligonucleotides may be synthesized directly on the solid support or
attached to the solid support subsequent to synthesis. Solid-supports
suitable for use in detection methods of the invention include substrates
made of silicon, glass, plastic, paper and the like, which may be formed,
for example, into wells (as in 96-well plates), slides, sheets,
membranes, fibers, chips, dishes, and beads. The solid support may be
treated, coated or derivatized to facilitate the immobilization of the
allele-specific oligonucleotide or target nucleic acid. For screening
purposes, hybridization probes of the polymorphic sequences may be used
where both forms are present, either in separate reactions, spatially
separated on a solid phase matrix, or labeled such that they can be
distinguished from each other.

[0055] Hybridization may also be performed with nucleic acid arrays and
subarrays such as described in WO 95/11995. The arrays would contain a
battery of allele-specific oligonucleotides representing each of the
polymorphic sites, wherein the spatial location of each oligonucleic acid
molecule is known. One or both polymorphic forms may be present in the
array, for example the polymorphism of position 1296 may be represented
by either, or both, of the listed nucleotides. Usually such an array will
include at least 2 different polymorphic sequences, i.e. polymorphisms
located at unique positions within the locus, and may include all of the
provided polymorphisms. Arrays of interest may further comprise
sequences, including polymorphisms, of other genetic sequences,
particularly other sequences of interest. The oligonucleotide sequence on
the array will usually be at least about 12 nt in length, may be the
length of the provided polymorphic sequences, or may extend into the
flanking regions to generate fragments of 100 to 200 nt in length. For
examples of arrays, see Ramsay (1998) Nat. Biotech. 16:4044; Hacia et al.
(1996) Nature Genetics 14:441-447; Lockhart et al. (1996) Nature
Biotechnol. 14:1675-1680; and De Risi et al. (1996) Nature Genetics
14:457-460.

[0057] A polymerase-mediated primer extension method may also be used to
identify the polymorphism(s). Several such methods have been described in
the patent and scientific literature and include the "Genetic Bit
Analysis" method (WO92/15712) and the ligase/polymerase mediated genetic
bit analysis (U.S. Pat. No. 5,679,524). Related methods are disclosed in
WO91/02087, WO90/09455, WO95/17676, U.S. Pat. Nos. 5,302,509, and
5,945,283. Extended primers containing a polymorphism may be detected by
mass spectrometry as described in U.S. Pat. No. 5,605,798. Another primer
extension method is allele-specific PCR (Ruao et al., Nucl. Acids Res.
17:8392, 1989; Ruao et al., Nucl. Acids Res. 19, 6877-6882, 1991; WO
93/22456; Turki et al., J. Clin. Invest. 95:1635-1641, 1995). In
addition, multiple polymorphic sites may be investigated by
simultaneously amplifying multiple regions of the nucleic acid using sets
of allele-specific primers as described in Wallace et al. (WO 89/10414).

[0058] A detectable label may be included in an amplification reaction.
Suitable labels include fluorochromes, e.g. fluorescein isothiocyanate
(FITC), rhodamine, Texas Red, phycoerythrin, allophycocyanin,
6-carboxyfluorescein (6-FAM),
2',7'-dimethoxy-4',5'-dichloro-6-carboxyfluorescein (JOE),
6-carboxy-X-rhodamine (ROX), 6-carboxy-2',4',7',4,7-hexachlorofluorescein
(HEX), 5-carboxyfluorescein (5-FAM) or
N,N,N',N'-tetramethy-6-carboxyrhodamine (TAMRA), radioactive labels, e.g.
.sup.32P, .sup.35S, .sup.3H; etc. The label may be a two stage system,
where the amplified DNA is conjugated to biotin, haptens, etc. having a
high affinity binding partner, e.g. avidin, specific antibodies, etc.,
where the binding partner is conjugated to a detectable label. The label
may be conjugated to one or both of the primers. Alternatively, the pool
of nucleotides used in the amplification is labeled, so as to incorporate
the label into the amplification product.

[0059] It is readily recognized by those ordinarily skilled in the art
that in order to maximize the signal to noise ratio, in probe
hybridization detection procedure, the polymorphic site should at the
center of the probe fragment used, whereby a mismatch has a maximum
effect on destabilizing the hybrid molecule; and in a PCR detection
procedure, the polymorphic site should be placed at the very 3'-end of
the primer, whereby a mismatch has the maximum effect on preventing a
chain elongation reaction by the DNA polymerase. The location of
nucleotides in a polynucleotide with respect to the center of the
polynucleotide are described herein in the following manner. When a
polynucleotide has an odd number of nucleotides, the nucleotide at an
equal distance from the 3' and 5' ends of the polynucleotide is
considered to be "at the center" of the polynucleotide, and any
nucleotide immediately adjacent to the nucleotide at the center, or the
nucleotide at the center itself is considered to be "within 1 nucleotide
of the center." With an odd number of nucleotides in a polynucleotide any
of the five nucleotides positions in the middle of the polynucleotide
would be considered to be within 2 nucleotides of the center, and so on.
When a polynucleotide has an even number of nucleotides, there would be a
bond and not a nucleotide at the center of the polynucleotide. Thus,
either of the two central nucleotides would be considered to be "within 1
nucleotide of the center" and any of the four nucleotides in the middle
of the polynucleotide would be considered to be "within 2 nucleotides of
the center," and so on.

[0060] In some embodiments, a composition contains two or more differently
labeled oligonucleotides for simultaneously probing the identity of
nucleotides or nucleotide pairs at two or more polymorphic sites. It is
also contemplated that primer compositions may contain two or more sets
of allele-specific primer pairs to allow simultaneous targeting and
amplification of two or more regions containing a polymorphic site.

[0061] Alternatively, the relevant portion of the gene of the sample of
interest may be amplified via PCR and directly sequenced, and the
sequence be compared to the wild type sequence shown in the figures. It
is readily recognized that, other than those disclosed specifically
herein, numerous primers can be devised to achieve the objectives. PCR
and sequencing techniques are well known in the art and reagents and
equipments are readily available commercially.

[0062] DNA markers have several advantages; segregation is easy to measure
and is unambiguous, and DNA markers are co-dominant, i.e., heterozygous
and homozygous animals can be distinctively identified. Once a marker
system is established selection decisions could be made very easily,
since DNA markers can be assayed any time after a blood sample can be
collected from the individual infant animal, or even earlier by testing
embryos in vitro if very early embryos are collected. The use of marker
assisted genetic selection will greatly facilitate and speed up cattle
breeding problems. For example, a modification of the multiple ovulation
and embryo transfer (MOET) procedure can be used with genetic marker
technology. Specifically, females are superovulated, eggs are collected,
in vitro fertilized using semen from superior males and implanted into
other females allowing for use of the superior genetics of the female (as
well as the male) without having to wait for her to give birth to one
calf at a time. Developing blastomeres at the 4-8 cell stage may be
assayed for presence of the marker, and selection decisions made
accordingly.

[0063] In one embodiment of the invention an assay is provided for
detection of presence of a desirable genotype using the markers.

[0064] The term "genotype" as used herein refers to the identity of the
alleles present in an individual or a sample. In the context of the
present invention a genotype preferably refers to the description of the
polymorphic alleles present in an individual or a sample. The term
"genotyping" a sample or an individual for a polymorphic marker refers to
determining the specific allele or the specific nucleotide carried by an
individual at a polymorphic marker.

[0065] The present invention is suitable for identifying a bovine,
including a young or adult bovine animal, an embryo, a semen sample, an
egg, a fertilized egg, or a zygote, or other cell or tissue sample
therefrom, to determine whether said bovine possesses the desired
genotypes of the present invention, some of which are indicative of
improved milk production traits.

[0066] Further provided is a method for genotyping one of the bovine genes
listed in Table 1, comprising determining for the two copies of the gene
present the identity of the nucleotide pair at the relevant SNP position.

[0067] One embodiment of a genotyping method of the invention involves
examining both copies of the gene, or a fragment thereof, to identify the
nucleotide pair at the polymorphic site in the two copies to assign a
genotype to the individual. In some embodiments, "examining a gene" may
include examining one or more of: DNA containing the gene, mRNA
transcripts thereof, or cDNA copies thereof. As will be readily
understood by the skilled artisan, the two "copies" of a gene, mRNA or
cDNA, or fragment thereof in an individual may be the same allele or may
be different alleles. In another embodiment, a genotyping method of the
invention comprises determining the identity of the nucleotide pair at
the polymorphic site.

[0068] The present invention further provides a kit for genotyping a
bovine sample, the kit comprising in a container a nucleic acid molecule,
as described above, designed for detecting the polymorphism, and
optionally at least another component for carrying out such detection.
Preferably, a kit comprises at least two oligonucleotides packaged in the
same or separate containers. The kit may also contain other components
such as hybridization buffer (where the oligonucleotides are to be used
as a probe) packaged in a separate container. Alternatively, where the
oligonucleotides are to be used to amplify a target region, the kit may
contain, preferably packaged in separate containers, a polymerase and a
reaction buffer optimized for primer extension mediated by the
polymerase, such as PCR.

[0069] In one embodiment the present invention provides a breeding method
whereby genotyping as described above is conducted on bovine embryos, and
based on the results, certain cattle are either selected or dropped out
of the breeding program.

[0070] Through use of the linked marker loci, procedures termed "marker
assisted selection" (MAS) may be used for genetic improvement within a
breeding nucleus; or "marker assisted introgression" for transferring
useful alleles from a resource population to a breeding nucleus (Soller
1990; Soller 1994).

[0071] The present invention discloses the association between the genes
listed in Table 1 and fertilization rate or embryonic survival.

[0072] The following examples are intended to illustrate preferred
embodiments of the invention and should not be interpreted to limit the
scope of the invention as defined in the claims.